| The research of the Group
is dedicated to understanding the cellular mechanisms that underlie changes
in cytoplasmic calcium concentration in general, and more specifically
those mechanisms which trigger contraction following an electrical signal
on the surface membrane of skeletal and cardiac muscle fibres. Several
different approaches are used to tackle this problem.
These include:
- electrophysiological studies of currents through single ion channel
proteins and of contraction in isolated bundles of intact muscle fibres
and in skinned segments of single fibres
- biochemical isolation and modification of ion channel proteins
- expression and mutation of ion channel proteins and proteins that
regulate the calcium release channels
- NMR and X-ray crystallographic studies of protein structure
The Ryanodine Receptor Calcium Release
Channel
The ubiquitous ryanodine receptor calcium release channel is found in
the membranes of intracellular calcium stores and is the major calcium
release pathway from these stores in many cell types. Although regulation
of cytoplasmic calcium concentrations is basic to the function of all
cells, the mechanisms controlling ryanodine receptor activity are not
well understood.
We are examining the regulation of calcium ion flow through the ryanodine
receptor by studying the currents through single channels incorporated
into artificial lipid bilayers. Our specific interests are the modulation
of channel activity by calcium and magnesium ions, following sulfhydryl
reduction and oxidation (by oxidants such as NO), by FK-506 binding proteins
(FKBPs), by co-proteins like triadin, junctin and calsequestrin and by
protein-protein interactions with the skeletal muscle L-type calcium channel
(an essential step in excitation-contraction coupling), which is also
known as a dihydropyridine receptor (DHPR). We have identified basic mechanisms
in (a) calcium magnesium regulation sites, (b) redox state, (c) FKBP and
Homer in controlling the "gating" of the ion channel. Our studies
have shown for the first time that small peptides, corresponding to a
sequence in the DHPR, both activate and inhibit single ryanodine receptor
channels, and that the activation is modified by FKBP12. We have further
shown how interactions between triadin, junctin and calsequestrin allow
the environment in the lumen of the SR to regulate Ca2+ release through
the RyR. We have also identified the triadin binding domain on the RyR
and shown an essential role for triadin in EC coupling. These studies
are continuing to identify regions of the RyR that bind to junctin and
regions on triadin and junctin that bind to the RyR. The research on the
RyR includes regions in the protein that are variably spliced, their role
in development, myotonic dystrophy and in excitation-contraction coupling.
Finally we are looking at interactions between glutathione transferases
and the cardiac ryanodine receptor and the possibility of using parts
of the GST protein as a template for drugs that will target the cardiac
ryanodine receptor in heart failure.
Future studies will investigate the sequences in the ryanodine receptor
and co-proteins, and the structural constraints that allow regulatory
interactions to proceed. We are also examining the effects of the ryanodine
receptor mutation in malignant hyperthermia on single channel activity.
Excitation Contraction Coupling
The Muscle Research Group was largely responsible for much of the basic
work on voltage-dependence of excitation-contraction coupling in mammalian
skeletal muscle. However, the molecular mechanism of excitation-contraction
coupling in skeletal muscle is still not properly understood. We know
that depolarization of the surface membrane activates a voltage sensor
which is a part of the dihydropyridine receptor in the transverse tubule
membrane. The loop between the second and third transmembrane segment
of the dihydropyridine receptor is thought to be involved in transmitting
the depolarisation-evoked signal to the ryanodine receptor. We have recently
published the first atomic level structure of the II-III loop and we are
examining the structure of a SPRY2 domain in the RyR that interacts with
the II-III loop. Future experiments will examine the interactions between
the II-III loop and activating peptides and other co-proteins, especially
the FKBPs, triadin and junctin, so that a model can be developed of the
in vivo activation of the ryanodine receptor by the dihydropyridine receptor
during excitation-contraction coupling.
Calsequestrin and heart disorders
Heart disease is the leading cause of death in Australia and is responsible
for over 50,000 deaths annually. Heart beat relies on the conversion of
an electrical signal originating in nervous tissue to the mechanical force
of synchronous heart beat. One of the quintessential steps in excitation
contraction coupling is the release of calcium ions from a calcium store
(the sarcoplasmic reticulum) located deep within the muscle cell. In a normally
functioning heart, the release of calcium from the sarcoplasmic reticulum
is under a myriad of control mechanisms and calcium homeostasis is thus
maintained.
Calsequestrin is the key calcium binding protein in the sarcoplasmic reticulum
of heart and in skeletal muscle. It is responsible for the stores high capacity
calcium binding and controls ryanodine receptor channel activity. Thus,
calsequestrin can regulate calcium release from the store by acting as a
sensor for the ryanodine receptor to monitor calcium concentration within
the store. The muscle research group are investigating the precise function
of calsequestrin in the heart and skeletal muscle and we were the first
group to identify calsequestrin as inhibitor of the ryanodine receptor.
We are also focused on how calcium mishandling by the cardiac sarcoplasmic
reticulum, resulting from severe toxicity (often induced by chemotherapeutic
agents) or mutations in CSQ, lead to several clinical disorders which result
in sudden death. |
